People are largely dependent upon fossil fuels for fulfilling our energy requirement. Bioalcohol, in particular ethanol is an attractive alternate transportation fuel to replace at least a part of petroleum. Fuels from renewable sources like agricultural and forest residues hold promise in reducing their dependence on fossil fuel without competing with food. The agricultural and forestry waste mostly consist of lignocellulose, which is made-up of highly structured cellulose surrounded by hemicellulose and lignin. In principle, it is possible to breakdown lignocellulose into the monosaccharides and ferment them into ethanol. However, cost associated with this process is a major hurdle in terms of commercial application. One of the key advancement in the economy of ethanol production from lignocellulosic biomass will be to efficiently ferment both hexose and pentose sugars released after hydrolysis of lignocellulose into ethanol.
Unfortunately, the conventional microorganisms used for ethanol fermentation, e.g., Saccharomyces cerevisiae and Zymomonas mobilis, do not have the capability to utilize pentose sugars, Attempts have been made to transfer genes for pentose degradation pathway from other organisms into S. cerevisiae and Z. mobilis. However, the disadvantages associated with foreign gene expression at large scale like instability, toxicity, containment, etc., prevent its wide usage. Escherichia coli, on the other hand, has the ability to ferment both hexose and pentose sugars and is being used to produce ethanol by various genetic manipulation The genetic manipulation of E. coli that does not involve introduction of foreign gene has been attempted with some successes and these technologies have advantages in the long-term genetic stability of the engineered strain.
Under anaerobic condition, E. coli produces ethanol through a pathway that involves pyruvate formate lyase (PFL), which converts pyruvate into acetyl CoA and formate (FIG. 1). However, this pathway is not redox balanced because in the process of metabolizing one mole of glucose into ethanol, four moles of NADH are consumed while only two moles of NADH are produced. This redox imbalance would negatively impact the yield of ethanol. However, there is an alternate pathway exists where converting glucose into ethanol or butanol is a redox balance process. Here pyruvate in converted into acetyl CoA and CO2 via pyruvate dehydrogenase complex (PDH) and in the process one molecule of NADH is produced. However, expression of PDH is repressed under anaerobic condition and remains active in the aerobically growing cells. To activate the expression of PDH under anaerobic condition, the promoter of PDH should be replaced with the one that is highly active under anaerobic condition. Attempts of replacing the PDH promoter with the PFL promoter have not been found to be very successful, as despite showing the enhanced expression of PDH under anaerobic condition and increased the yield of ethanol, the net ethanol productivity was low.
Thus, there is a need for a modified bacterial strain with genetic alterations that can ferment both hexose and pentose sugars in order to produce bioalcohol such as ethanol with high productivity.